Similar to the powerful endogenous cardioprotective mechanism of ischemic preconditioning, anesthetic-induced preconditioning (APC) has emerged as an equally effective cardioprotective intervention with better risk-to-benefit ratio for the patient. During the current cycle of the Program Project we have identified key elements and mechanisms involved in APC. Central to cardioprotection is the knowledge we have gained regarding the regulation of mitochondrial function by volatile anesthetics. Given the fundamental role of mitochondria in myocardial energetics and oxidative stress, we believe that they are a promising target for protective strategies such as APC. In contrast, disease states resistant to APC (e.g. diabetes) contain fundamental disturbances of mitochondrial function. The central theme of this Program Project is to elucidate the molecular mechanisms underlying APC. Specifically, we hypothesize that attenuation of permeability transition (PT) pore opening after ischemia and reperfusion is central to many of the phenotypic differences observed after exposure to volatile anesthetics. This Program will consist of three interrelated research projects supported by two Cores. Project I (Warltier) will focus on defining the temporal sequence of activation of key cardioprotective proteins related to the regulation of NO production via HIF1a-VEGF-NO axis by volatile anesthetics. Disruption of these elements and their effect on sarcKATp channel activity, mitochondrial-derived ROS formation, and tissue and cell injury will be determined. Project II (Bosnjak) will elucidate mechanisms involved in volatile anesthetic-dependent modulation of PT pore opening, a critical end effector of APC. It will address several factors that are critical to the role of the PT pore in APC such as mitochondrial bioenergetics and its proteome and the contribution of sarcKATp channels. Computational models of mitochondrial bioenergetics will be used to test specific hypotheses related to the effects of volatile anesthetics. Project III (Kersten) will investigate mechanisms involved in the attenuation of APC in diabetic animals. It will exploit a novel rat model of type 2 diabetes in which we were able to selectively switch the mitochondrial genome to further dissect the role of mitochondria and eNOS-sensitive pathway during impaired APC. All three Projects will be supported by a Biochemical and Molecular Biology Core (Harder) and a Proteomics Core (Olivier). These Cores will provide state-ofthe- art techniques in gene silencing, real time PCR, mitochondrial proteome, cell cultures, mitochondrial function assays, confocal microscopy and pathology. This Program Project represents a comprehensive effort to leverage our existing infrastructure and programmatic experience in physiology, biophysics, genomics, proteomics, and computational biology to advance our understanding of the cellular and subcellular effects of anesthetics in organ protection. Our findings are likely to have a significant impact in the clinical use of volatile anesthetics.